To investigate whether there exits the dose- and time- dependent effect of RNA interference(RNAi) when the introduced extraneous reporter gene was suppressed by RNAi in mammalian cell lines.The expression vectors carrying reporter component were cotransfected with the plasmids coding short hairpin RNAs(shRNAs) into HEK293H cell using lipofectamine 2000 reagent, and the consequent inhibitory effect mediated by RNA interference was observed. After transfection, the transient expression of shRNAs could specifically inhibit the extraneous reporter in mammalian cell.The expression of mRNA and protein of enhanced green fluorescent protein(EGFP)gene was determined at 12, 24, 48, 60, 72, 96 h after transfection in HEK293 cell. The results showed that the decrease of EGFP mRNA or protein level was not obvious at 12 h, but gradually became more evident during from 24 to 48 h.The decrease achieved the maximal degree during from 48 to 72 h, then became weakened and restored subsequently. It indicated that the effect of RNA interference underwent a tendency of from weak to strong, then from strong to weak, and ultimately disappeared gradually. The efficiency of inhibition caused by RNAi was related to the dose of RNA interfering vector within a confined limit in HEK293H cell cotransfected with a series of dose-proportional vectors as pd1EGFP and psh-d1EGFP, whereas it nearly kept constant when the dose of interfering plasmid was sufficient to depress the expression of extraneous genes. Simultaneously, the variation of luciferase activity also displayed the similar effect when its expression was suppressed by RNAi in HEK293H or HeLa cell.. It concluded that vector-based RNA interference took on time- and dose- dependent effect in mammalian cell, which provides certain theoretical reference or valuable clue to the utility of RNAi.